REGULATION OF DNA-BINDING AND TRANSACTIVATION IN P53 BY NUCLEAR-LOCALIZATION AND PHOSPHORYLATION

Compelling evidence indicates that p53 acts as a transcription factor and that this activity is regulated by several factors including subce llular localization and phosphorylation status of the protein. To lear n more about how these two processes determine whether p53 becomes act ivated, we studied the temperature sensitive murine p53, p53(val135). At nonpermissive temperatures, p53(val135) remains sequestered in the cytoplasm of cells ,which express it. Electrophoretic mobility shift a ssays demonstrated that, under these conditions, the protein lacked DN A binding activity. However, by shifting to the permissive temperature , p53(val135) became concentrated in the nucleus, hyperphosphorylated, and had acquired the ability to bind DNA in a sequence specific manne r. This was accompanied by the induction of two p53 regulated genes, m dm2 and p21(waf1), which indicated that p53(val135) had become an acti ve transcription factor. Two dimensional gel electrophoresis and trypt ic peptide mapping showed that entry into the nucleus resulted in the appearance of new phosphorylated isoforms and that the protein had bec ome extensively phosphorylation at the N-terminus. Notably, phosphoryl ation at the N-terminus occurred only in the nucleus, whereas phosphor ylation at the C-terminus could occur in both the cytoplasm and the nu cleus. Based on these observations, we suggest that phosphorylation of p53's N-terminus is compartmentally restricted.